Difference between revisions of "Part:BBa K4765121"

From left lane(1) to right lane(3) indicate the successful construction of galU, galU + pgmA, and galU + pgmA + mScarlet.

We constructed BBa_K4765121 and BBa_K4765122 into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represent the protein expression of BBa_K4765121, Lane 3 to 4 represent the protein expression of BBa_K4765122. Each part has one leaky expression version and one induced version. As indicated by the red arrow, we successfully expressed proteins in BBa_K4765121 and BBa_K4765122.

Aggregation Assay and Cluster Forming

We found EPS-expressing bacteria are "heavier" and precipitate faster, likely due to being more "sticky".

To confirm if the E. coli expressing EPS became more “sticker”, we allowed the bacterial suspension to settle for a period and observed its aggregation process. As is shown in Figure 3, EPS-expressing E. coli exhibited significantly faster aggregation compared to the control group, indicating that EPS-expression bacteria is "sticker", leading to bacterial aggregation.

Additionally, we mixed the E. coli expressing EPS with bacteria only expressing StayGold before loading into the flow chamber, and fluorescence microscopy imaging subsequently. As is shown in Figure 4, Bacteria expressing EPS, indicated by red fluorescence, tend to cluster together, while the control group bacteria with green fluorescence are sparsely distributed. This suggests that EPS-expressing bacteria are "stickier" and have the capability to form clusters.

Figure 3. Aggregation Experiment Results The four on the left are EPS-expressing E. coli, while the yellow one on the right represents the control group with E. coli only expressing stayGold.

Figure 4. Fluorescence Microscopy Imaging (150x) of Cluster Forming Red fluorescence: EPS-expressing E. coli, Green fluorescence: E. coli only expresses StayGold.

Adhesion Assay

To validate the adhesion effects of EPS, we performed microscopy imaging using a chamber-based approach. After mixing the E.coli expressing EPS with an appropriate concentration of PDL (Poly-D-Lysine), it was forcefully pipetted ten times before being loaded into the flow chamber. Subsequently, we conducted fluorescence microscopy imaging. We applied two different force intensities to wash the adherent bacteria and observed whether the EPS-expressing E.coli could remain adhered to the glass slide without being dislodged.

As is shown in Figure 5, the two left images under lower-speed washing, EPS-expressing E. coli cells (red fluorescence indicated by white arrows) maintained adhesion to the glass surface for an extended period without being dislodged, while the control group (green fluorescence) was washed away. In the two right images, subjecting adherent E. coli to more intense washing removed non-adherent cells completely. However, EPS-expressing E. coli cells (white arrows) could still adhere to the glass surface for a considerable time. This indicates that EPS effectively promotes E. coli adhesion.

Figure 5. Fluorescence Microscopy Imaging (150x) of E.coli Adhesion All four images were captured from the same field of view, showing E.coli adhesion under different washing conditions. The two images on the left depict bacterial adhesion over an extended period under mild washing, while the two images on the right show bacterial adhesion still remains under stronger washing.

In the following video, we further demonstrated the adhesion process of EPS-expressing E. coli cells (red fluorescence indicated by white arrows) during the aforementioned washing procedures.

Figure 6. Visualization of Escherichia coli Adhesion through Fluorescence Microscopy Magnification: 150x. Video Duration: Captured at 200ms intervals

Sequence and Features

References

1. ↑ Levander, F., Svensson, M., & Rådström, P. (2002). Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus. Applied and Environmental Microbiology, 68(2), 784–790. https://doi.org/10.1128/AEM.68.2.784-790.2002